There is a critical clinical need for a noninvasive high-resolution imaging technology for early cancer detection and guidance of biopsy in internal organs. Two-photon fluorescence (TPF) and second harmonic generation (SHG) microscopy is a powerful technology to address the above clinical need by providing structural and biochemical/metabolic information about biological tissues at subcellular resolution without the need for tissue removal or external fluorescent agents. However, its in vivo clinical application remains extremely limited due to the lack of a miniature technology platform. The objective of this multidisciplinary proposal is to develop an all-fiber-optic scanning endomicroscopy technology which is able to bring TPF/SHG microscopy to clinic for internal organ imaging. It involves 5 partners with 2 from academia and 3 from industry. The proposed technology will integrate all essential functions of a scanning laser microscope into a single flexible fiber-optic probe of a small diameter (~2.4-3.4 mm), with built-in mechanisms for femtosecond pulse delivery, dispersion management, nonlinear effect suppression, beam focusing, rapid 2D raster beam scanning, TPF/SHG collection, and focus tracking (or depth scanning). The small size permits its integration with a standard red-flagging technology (such as a gastroscope). In this proposal, we plan to tackle the major challenges in developing such an endomicroscopy technology and evaluate its feasibility for subcellular resolution imaging and for cancer detection. The Specific Aims are to: (1) Develop new double-clad fibers (DCF) of a pure silica core, large inner clad and numerical aperture to dramatically suppress the in-fiber TPF/SHG background (e.g. by 50 folds) and improve TPF/SHG collection efficiency (e.g. by ~15 folds) over commercially available DCFs; (2) Develop a super-achromatic microlens of a 2.1mm diameter and 0.6 NA to improve the TPF/SHG collection efficiency by at least 20 folds over a GRIN lens; (3) Explore a novel approach based on high-order mode DCFs to suppress nonlinear effects in optic fiber and improve TPF/SHG excitation probability; (4) Develop novel MEMS scanners of a small footprint (1.6 x 1.6 mm) and an extremely low drive voltage (10V max) to achieve rapid 2D raster beam scanning and real-time focus tracking. A fully integrated endomicroscope with customized DCFs, microlens and MEMS scanners will be developed, capable of 3D TPF/SHG imaging; (5) Conduct in vivo endoscopic TPF/SHG imaging of swine esophagus to evaluate the performance, and design, engineering and operation issues of the scanning probe; and (6) Evaluate the feasibility of the proposed technology for cancer detection and tumor margin identification using ex vivo human esophagus specimens, and correlate TPF/SHG endomicroscopy images with corresponding histology. The significance of the proposed research is to translate the powerful TPF/SHG microscopy technology to clinical practice for cancer detection and image-guided biopsy in internal organs, and enable noninvasive real-time visualization of tissue histopathology in situ to significantly improve diagnostic and biopsy yields. In addition, the proposed endomicroscopy technology will also be applicable (although outside the scope of this proposal) to many other clinical scenarios such as for guidance of surgical interventions and for in vivo assessment of metabolic function of living tissues.